Methods for detecting chromogranin a by mass spectrometry

ABSTRACT

Provided are methods for detecting chromogranin A by mass spectrometry. In another aspect, provided herein are methods for quantitating chromogranin A by mass spectrometry. In another aspect, provided herein are methods for prognosis of or measuring the size of neuroendocrine tumors by mass spectrometry.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims benefit of U.S. Provisional Application No.62/644,210, filed Mar. 16, 2018, which is incorporated by referenceherein in its entirety.

BACKGROUND OF THE INVENTION

Chromogranin-A (CgA) is a 50 kDa acidic glycoprotein expressed in thesecretory granules of neuroendocrine tissue. Currently, blood levels ofCgA are primarily measured using various immunoassays. However, as withany antibody-based assay, limitations arising from non-specific bindingand a reduced dynamic range requiring sample dilution, pose hurdles inimplementing such tests in diagnostic laboratories.

An accurate and sensitive assay for quantitating chromogranin A isneeded.

SUMMARY OF THE INVENTION

Provided herein are methods for detecting or determining the amount ofchromogranin A (CgA) in a sample by mass spectrometry, including tandemmass spectrometry.

In certain embodiments, the methods provided herein are for detecting ordetermining the amount of chromogranin A (CgA) comprising (a) purifyingCgA in the sample; (b) ionizing CgA to produce ions detectable by massspectrometry; and (c) detecting or determining the amount of the CgAion(s) by mass spectrometry; wherein the amount of the CgA ion(s) isrelated to the amount of CgA in the sample.

In certain embodiments, the methods provided herein are for detecting ordetermining the amount of chromogranin A (CgA) comprising (a) subjectingthe sample to solid phase extraction; (b) enzymatically digesting theCgA; (c) subjecting the CgA to liquid chromatography; (d) ionizing CgAto produce ions detectable by mass spectrometry; and (e) detecting ordetermining the amount of the CgA ion(s) by mass spectrometry; whereinthe amount of the CgA ion(s) is related to the amount of CgA in thesample.

In certain embodiments, methods provided herein comprise selectedreaction monitoring (SRM) mass spectrometry.

In some embodiments, the methods provided herein are fully automated.

In some embodiments, the methods provided herein are antibody-freemethods.

In some embodiments, purifying provided herein comprises extraction ofserum using solid phase extraction (SPE). In some embodiments, SPE is ananion exchange solid-phase extraction. In some embodiments, SPE is amixed-mode anion exchange solid-phase extraction. In some embodiments,extracted samples are concentrated.

In some embodiments, purifying provided herein comprises liquidchromatography. In some embodiments, the liquid chromatography compriseshigh performance liquid chromatography (HPLC). In some embodiments, theliquid chromatography comprises high turbulence liquid chromatography(HTLC).

In some embodiments, extracted samples are enzymatically digested. Insome embodiments, extracted samples are enzymatically digested bytrypsin.

In some embodiments, the ionization comprises electrospray ionization(ESI). In some embodiments, the ionization comprises ionizing inpositive mode. In some embodiments, the ionization comprises ionizing innegative mode.

In some embodiments, the ionization comprises atmospheric pressurechemical ionization (APCI). In some embodiments, the ionizationcomprises ionizing in positive mode. In some embodiments, the ionizationcomprises ionizing in negative mode.

In some embodiments, methods provided herein comprise measuring theamount of precursor ion having a mass-to-charge ratio of 593.2±0.5.

In some embodiments, methods provided herein comprise measuring theamount of precursor ion having a mass-to-charge ratio of 729.6±0.5.

In some embodiments, methods provided herein comprise measuring theamount of a fragment of chromogranin A. In some embodiments, the CgAfragment measured comprises a sequence ELQDLALQGA (SEQ ID NO:1). In someembodiments, the CgA fragment measured comprises a sequenceRRPEDQELESLSAIEAELEK (SEQ ID NO:4).

In some embodiments, methods provided herein comprise measuring theamount of fragment ion having a mass-to-charge ratio of 516.3±0.5 or815.5±0.5 or both.

In some embodiments, methods provided herein comprise measuring theamount of fragment ion having a mass-to-charge ratio of 831.5±0.5 or989.5±0.5 or both.

In some embodiments, methods provided herein further comprise adding aninternal standard. In some embodiments, the internal standard isisotopically labeled. In some embodiments, the internal standardcomprises C¹³N¹⁵ labeled amino acids. In some embodiments, the internalstandard is labeled on a leucine (L) or lysine (K). In some embodiments,the internal standard comprises a sequenceILSILRHQNLLKELQDLAL*QGAK*ERAHQQK (SEQ ID NO:2), wherein * is a C¹³N¹⁵labeled amino acid. In some embodiments, the internal standard comprisesa sequence RRPEDQELESL*SAIEAELEK* (SEQ ID NO:5), wherein * is a C¹³N¹⁵labeled amino acid.

In some embodiments, methods provided herein comprise measuring theamount of internal standard precursor ion having a mass-to-charge ratioof 600.8±0.5 and/or product ion having a mass-to-charge ratio of602.4±0.5, 830.6±0.5, or 958.7±0.5.

In some embodiments, methods provided herein comprise measuring theamount of internal standard precursor ion having a mass-to-charge ratioof 600.8±0.5 and/or product ion having a mass-to-charge ratio of734.6±0.5, 839.5±0.5, or 997.6±0.5.

In certain embodiments, the limit of quantitation of the methods is lessthan or equal to 100 ng/mL. In some embodiments, the limit ofquantitation of the methods is less than or equal to 90 ng/mL. In someembodiments, the limit of quantitation of the methods is less than orequal to 80 ng/mL. In some embodiments, the limit of quantitation of themethods is less than or equal to 70 ng/mL. In some embodiments, thelimit of quantitation of the methods is less than or equal to 60 ng/mL.In some embodiments, the limit of quantitation of the methods is lessthan or equal to 50 ng/mL.

In some embodiments, the limit of detection of the methods is less thanor equal to 50 ng/mL. In some embodiments, the limit of detection of themethods is less than or equal to 40 ng/mL. In some embodiments, thelimit of detection of the methods is less than or equal to 35.5 ng/mL.

In some embodiments, methods provided herein comprise linearity ofquantitation across a range between 50 ng/mL to 50,000 ng/mL.

In some embodiments, methods provided herein comprise inter- andintra-assay reproducibility of CV≤15%.

In some embodiments, CgA is not derivatized prior to mass spectrometry.

In certain embodiments, the sample is a body fluid. In some embodiments,the sample is cerebrospinal fluid (CSF). In some embodiments, the sampleis plasma or serum. In some embodiments, the sample is whole blood. Insome embodiments, the sample is saliva or urine.

In some embodiments, the methods may include adding an agent to thesample in an amount sufficient to deproteinate the sample.

In some embodiments, elevated levels of chromogranin A as compared to areference range indicates increased risk of neuroendocrine tumors (NET).In some embodiments, quantitated levels of chromogranin A indicates thesize of the neuroendocrine tumor. In some embodiments, quantitatedlevels of chromogranin A indicates the tumor burden of theneuroendocrine tumor. In some embodiments, quantitated levels ofchromogranin A indicates the response to treatment of the neuroendocrinetumor. In some embodiments, quantitated levels of chromogranin Aindicates the prognosis of the neuroendocrine tumor.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “aprotein” includes a plurality of protein molecules.

As used herein, the term “purification” or “purifying” does not refer toremoving all materials from the sample other than the analyte(s) ofinterest. Instead, purification refers to a procedure that enriches theamount of one or more analytes of interest relative to other componentsin the sample that may interfere with detection of the analyte ofinterest. Samples are purified herein by various means to allow removalof one or more interfering substances, e.g., one or more substances thatwould interfere with the detection of selected CgA parent and daughterions by mass spectrometry.

As used herein, the term “test sample” refers to any sample that maycontain CgA. As used herein, the term “body fluid” means any fluid thatcan be isolated from the body of an individual. For example, “bodyfluid” may include blood, plasma, serum, bile, saliva, urine, tears,perspiration, and the like.

As used herein, the term “derivatizing” means reacting two molecules toform a new molecule. Derivatizing agents may include isothiocyanategroups, dinitro-fluorophenyl groups, nitrophenoxycarbonyl groups, and/orphthalaldehyde groups, and the like.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of “liquidchromatography” include reverse phase liquid chromatography (RPLC), highperformance liquid chromatography (HPLC), and high turbulence liquidchromatography (HTLC).

As used herein, the term “high performance liquid chromatography” or“HPLC” refers to liquid chromatography in which the degree of separationis increased by forcing the mobile phase under pressure through astationary phase, typically a densely packed column.

As used herein, the term “high turbulence liquid chromatography” or“HTLC” refers to a form of chromatography that utilizes turbulent flowof the material being assayed through the column packing as the basisfor performing the separation. HTLC has been applied in the preparationof samples containing two unnamed drugs prior to analysis by massspectrometry. See, e.g., Zimmer et al., J. Chromatogr A. 854: 23-35(1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368, 5,795,469, and5,772,874, which further explain HTLC. Persons of ordinary skill in theart understand “turbulent flow”. When fluid flows slowly and smoothly,the flow is called “laminar flow”. For example, fluid moving through anHPLC column at low flow rates is laminar. In laminar flow the motion ofthe particles of fluid is orderly with particles moving generally instraight lines. At faster velocities, the inertia of the water overcomesfluid frictional forces and turbulent flow results. Fluid not in contactwith the irregular boundary “outruns” that which is slowed by frictionor deflected by an uneven surface. When a fluid is flowing turbulently,it flows in eddies and whirls (or vortices), with more “drag” than whenthe flow is laminar. Many references are available for assisting indetermining when fluid flow is laminar or turbulent (e.g., TurbulentFlow Analysis: Measurement and Prediction, P. S. Bernard & J. M.Wallace, John Wiley & Sons, Inc., (2000); An Introduction to TurbulentFlow, Jean Mathieu & Julian Scott, Cambridge University Press (2001)).

As used herein, the term “gas chromatography” or “GC” refers tochromatography in which the sample mixture is vaporized and injectedinto a stream of carrier gas (as nitrogen or helium) moving through acolumn containing a stationary phase composed of a liquid or aparticulate solid and is separated into its component compoundsaccording to the affinity of the compounds for the stationary phase.

As used herein, the term “large particle column” or “extraction column”refers to a chromatography column containing an average particlediameter greater than about 35 μm. As used in this context, the term“about” means ±10%. In a preferred embodiment the column containsparticles of about 60 μm in diameter.

As used herein, the term “analytical column” refers to a chromatographycolumn having sufficient chromatographic plates to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. Such columnsare often distinguished from “extraction columns”, which have thegeneral purpose of separating or extracting retained material fromnon-retained materials in order to obtain a purified sample for furtheranalysis. As used in this context, the term “about” means ±10%. In apreferred embodiment the analytical column contains particles of about 4μm in diameter.

As used herein, the term “on-line” or “inline”, for example as used in“on-line automated fashion” or “on-line extraction” refers to aprocedure performed without the need for operator intervention. Incontrast, the term “off-line” as used herein refers to a procedurerequiring manual intervention of an operator. Thus, if samples aresubjected to precipitation, and the supernatants are then manuallyloaded into an autosampler, the precipitation and loading steps areoff-line from the subsequent steps. In various embodiments of themethods, one or more steps may be performed in an on-line automatedfashion.

As used herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z”. MS technology generally includes (1)ionizing the compounds to form charged compounds; and (2) detecting themolecular weight of the charged compounds and calculating amass-to-charge ratio. The compounds may be ionized and detected by anysuitable means. A “mass spectrometer” generally includes an ionizer andan ion detector. In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrographic instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”). See, e.g., U.S. Pat. No. 6,204,500,entitled “Mass Spectrometry From Surfaces;” U.S. Pat. No. 6,107,623,entitled “Methods and Apparatus for Tandem Mass Spectrometry;” U.S. Pat.No. 6,268,144, entitled “DNA Diagnostics Based On Mass Spectrometry;”U.S. Pat. No. 6,124,137, entitled “Surface-Enhanced PhotolabileAttachment And Release For Desorption And Detection Of Analytes;” Wrightet al., Prostate Cancer and Prostatic Diseases 2:264-76 (1999); andMerchant and Weinberger, Electrophoresis 21:1164-67 (2000).

As used herein, the term “operating in negative ion mode” refers tothose mass spectrometry methods where negative ions are generated anddetected. The term “operating in positive ion mode” as used herein,refers to those mass spectrometry methods where positive ions aregenerated and detected.

As used herein, the term “ionization” or “ionizing” refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those having anet negative charge of one or more electron units, while positive ionsare those having a net positive charge of one or more electron units.

As used herein, the term “electron ionization” or “EI” refers to methodsin which an analyte of interest in a gaseous or vapor phase interactswith a flow of electrons. Impact of the electrons with the analyteproduces analyte ions, which may then be subjected to a massspectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methodsin which a reagent gas (e.g. ammonia) is subjected to electron impact,and analyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers tomethods in which a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, andtriethanolamine. The choice of an appropriate matrix for a compound orsample is an empirical process.

As used herein, the term “matrix-assisted laser desorption ionization”or “MALDI” refers to methods in which a non-volatile sample is exposedto laser irradiation, which desorbs and ionizes analytes in the sampleby various ionization pathways, including photoionization, protonation,deprotonation, and cluster decay. For MALDI, the sample is mixed with anenergy-absorbing matrix, which facilitates desorption of analytemolecules.

As used herein, the term “surface enhanced laser desorption ionization”or “SELDI” refers to another method in which a non-volatile sample isexposed to laser irradiation, which desorbs and ionizes analytes in thesample by various ionization pathways, including photoionization,protonation, deprotonation, and cluster decay. For SELDI, the sample istypically bound to a surface that preferentially retains one or moreanalytes of interest. As in MALDI, this process may also employ anenergy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI,” refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Solution reaching the end of the tube is vaporized(nebulized) into a jet or spray of very small droplets of solution insolvent vapor. This mist of droplets flows through an evaporationchamber, which is heated slightly to prevent condensation and toevaporate solvent. As the droplets get smaller the electrical surfacecharge density increases until such time that the natural repulsionbetween like charges causes ions as well as neutral molecules to bereleased.

As used herein, the term “atmospheric pressure chemical ionization” or“APCI,” refers to mass spectroscopy methods that are similar to ESI;however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then ions are typically extracted into the mass analyzer by use of a setof differentially pumped skimmer stages. A counterflow of dry andpreheated N₂ gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESI foranalyzing less-polar species.

The term “Atmospheric Pressure Photoionization” or “APPI” as used hereinrefers to the form of mass spectroscopy where the mechanism for thephotoionization of molecule M is photon absorption and electron ejectionto form the molecular ion M+. Because the photon energy typically isjust above the ionization potential, the molecular ion is lesssusceptible to dissociation. In many cases it may be possible to analyzesamples without the need for chromatography, thus saving significanttime and expense. In the presence of water vapor or protic solvents, themolecular ion can extract H to form MH+. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M+ and MH+ is constant. Drug compounds in protic solvents areusually observed as MH+, whereas nonpolar compounds such as naphthaleneor testosterone usually form M+. Robb, D. B., Covey, T. R. and Bruins,A. P. (2000): See, e.g., Robb et al., Atmospheric pressurephotoionization: An ionization method for liquid chromatography-massspectrometry. Anal. Chem. 72(15): 3653-3659.

As used herein, the term “inductively coupled plasma” or “ICP” refers tomethods in which a sample interacts with a partially ionized gas at asufficiently high temperature such that most elements are atomized andionized.

As used herein, the term “field desorption” refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

As used herein, the term “desorption” refers to the removal of ananalyte from a surface and/or the entry of an analyte into a gaseousphase.

As used herein, the term “limit of quantification”, “limit ofquantitation” or “LOQ” refers to the point where measurements becomequantitatively meaningful. The analyte response at this LOQ isidentifiable, discrete and reproducible with a precision of 20% and anaccuracy of 80% to 120%.

As used herein, the term “limit of detection” or “LOD” is the point atwhich the measured value is larger than the uncertainty associated withit. The LOD is defined arbitrarily as 2 standard deviations (SD) fromthe zero concentration.

As used herein, an “amount” of CgA in a body fluid sample refersgenerally to an absolute value reflecting the mass of CgA detectable involume of body fluid. However, an amount also contemplates a relativeamount in comparison to another CgA amount. For example, an amount ofCgA in a body fluid can be an amount which is greater than or less thana control or normal level of CgA normally present.

The term “about” as used herein in reference to quantitativemeasurements not including the measurement of the mass of an ion, refersto the indicated value plus or minus 10%. Mass spectrometry instrumentscan vary slightly in determining the mass of a given analyte. The term“about” in the context of the mass of an ion or the mass/charge ratio ofan ion refers to +/−0.5 atomic mass unit.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the analytical workflow for the CgA LC-MS/MS assay.Negatively charged CgA binds through electrostatic, lipophilic, andhydrophilic interactions with the mixed-mode anion exchange resin underconditions where it is retained while other proteins are washed away.After elution, the isolated CgA is digested with trypsin and arepresentative peptide is quantified by LC-MS/MS analysis.

FIG. 2 shows three calibration curves run over three separate days.Linear range was demonstrated to be 50 to 50,000 ng/mL. CV was less than10%.

FIG. 3 shows chromatograms corresponding to patient specimens withnormal (top) and high (bottom) CgA values.

FIG. 4 shows average ELISA immunoassay values as compared to the valuesobtained by LC-MS/MS.

FIG. 5 shows a comparison between CisBio immunoassay and LC-MS/MS CgAassay (Passing & Bablok curve fit, 308 samples).

FIG. 6 shows peak area graphs for normal and abnormal CgA levelsdetermined by LC-MS/MS.

FIGS. 7A and B show an example chromatogram for chromogranin internalstandard.

DETAILED DESCRIPTION OF THE INVENTION

Levels of CgA are increased in the presence of neuroendocrine-derivedtumors (NET), making CgA useful serum marker for monitoring patientswith NETs. Circulating levels of serum CgA are proportional to tumorburden, providing prognostic information in treatment response. Here wedescribe a novel, fully automated, antibody-free LC-MS/MS assay toquantitate chromogranin-A out of serum. Exploiting the acidic propertiesof CgA, 100 uL of serum is extracted using an anion exchange solid-phaseextraction plate followed by addition of internal standard. Theextracted sample is then concentrated and enzymatically digested usingtrypsin. A unique peptide to CgA is then chromatographically resolvedand analyzed by SRM on a Sciex 6500+ QTrap. The ratio of the analytepeak area to the isotopically labeled internal standard peak area isused to achieve quantitation. CgA shows linearity across a wide range(50-50000 ng/mL, R²≥0.99), as well as inter- and intra-assayreproducibility (CV≤15%). A cohort of 300 patient samples was analyzedto compare CgA serum values measured by the Cisbio CGA-ELISA-USimmunoassay to the described LC-MS/MS assay. When comparing immunoassayand LC-MS/MS measurements for CgA in this cohort, an R² of 0.71 wasobserved, showing a good correlation between the two assay platforms.

In certain embodiments, the methods provided herein are for detecting ordetermining the amount of chromogranin A (CgA) comprising (a) purifyingCgA in the sample; (b) ionizing CgA to produce ions detectable by massspectrometry; and (c) detecting or determining the amount of the CgAion(s) by mass spectrometry; wherein the amount of the CgA ion(s) isrelated to the amount of CgA in the sample.

In certain embodiments, the methods provided herein are for detecting ordetermining the amount of chromogranin A (CgA) comprising (a) subjectingthe sample to solid phase extraction; (b) enzymatically digesting theCgA; (c) subjecting the CgA to liquid chromatography; (d) ionizing CgAto produce ions detectable by mass spectrometry; and (e) detecting ordetermining the amount of the CgA ion(s) by mass spectrometry; whereinthe amount of the CgA ion(s) is related to the amount of CgA in thesample.

In certain embodiments, methods provided herein comprise selectedreaction monitoring (SRM) mass spectrometry.

In some embodiments, the methods provided herein are fully automated.

In some embodiments, the methods provided herein are antibody-freemethods.

In some embodiments, purifying provided herein comprises extraction ofserum using solid phase extraction (SPE). In some embodiments, SPE is ananion exchange solid-phase extraction. In some embodiments, SPE is amixed-mode anion exchange solid-phase extraction. In some embodiments,extracted samples are concentrated.

In some embodiments, purifying provided herein comprises liquidchromatography. In some embodiments, the liquid chromatography compriseshigh performance liquid chromatography (HPLC). In some embodiments, theliquid chromatography comprises high turbulence liquid chromatography(HTLC).

In some embodiments, extracted samples are enzymatically digested. Insome embodiments, extracted samples are enzymatically digested bytrypsin.

In some embodiments, the ionization comprises electrospray ionization(ESI). In some embodiments, the ionization comprises ionizing inpositive mode. In some embodiments, the ionization comprises ionizing innegative mode.

In some embodiments, the ionization comprises atmospheric pressurechemical ionization (APCI). In some embodiments, the ionizationcomprises ionizing in positive mode. In some embodiments, the ionizationcomprises ionizing in negative mode.

In some embodiments, methods provided herein comprise measuring theamount of precursor ion having a mass-to-charge ratio of 593.2±0.5.

In some embodiments, methods provided herein comprise measuring theamount of precursor ion having a mass-to-charge ratio of 729.6±0.5.

In some embodiments, methods provided herein comprise measuring theamount of a fragment of chromogranin A. In some embodiments, the CgAfragment measured comprises a sequence ELQDLALQGA (SEQ ID NO:1). In someembodiments, the CgA fragment measured comprises a sequenceRRPEDQELESLSAIEAELEK (SEQ ID NO:4).

In some embodiments, methods provided herein comprise measuring theamount of fragment ion having a mass-to-charge ratio of 516.3±0.5 or815.5±0.5 or both.

In some embodiments, methods provided herein comprise measuring theamount of fragment ion having a mass-to-charge ratio of 831.5±0.5 or989.5±0.5 or both.

In some embodiments, methods provided herein further comprise adding aninternal standard. In some embodiments, the internal standard isisotopically labeled. In some embodiments, the internal standardcomprises C¹³N¹⁵ labeled amino acids. In some embodiments, the internalstandard is labeled on a leucine (L) or lysine (K). In some embodiments,the internal standard comprises a sequenceILSILRHQNLLKELQDLAL*QGAK*ERAHQQK (SEQ ID NO:2), wherein * is a C¹³N¹⁵labeled amino acid. In some embodiments, the internal standard comprisesa sequence RRPEDQELESL*SAIEAELEK* (SEQ ID NO:5), wherein * is a C¹³N¹⁵labeled amino acid.

In some embodiments, methods provided herein comprise measuring theamount of internal standard precursor ion having a mass-to-charge ratioof 600.8±0.5 and/or product ion having a mass-to-charge ratio of602.4±0.5, 830.6±0.5, or 958.7±0.5.

In some embodiments, methods provided herein comprise measuring theamount of internal standard precursor ion having a mass-to-charge ratioof 600.8±0.5 and/or product ion having a mass-to-charge ratio of734.6±0.5, 839.5±0.5, or 997.6±0.5.

In certain embodiments, the limit of quantitation of the methods is lessthan or equal to 100 ng/mL. In some embodiments, the limit ofquantitation of the methods is less than or equal to 90 ng/mL. In someembodiments, the limit of quantitation of the methods is less than orequal to 80 ng/mL. In some embodiments, the limit of quantitation of themethods is less than or equal to 70 ng/mL. In some embodiments, thelimit of quantitation of the methods is less than or equal to 60 ng/mL.In some embodiments, the limit of quantitation of the methods is lessthan or equal to 50 ng/mL.

In some embodiments, the limit of detection of the methods is less thanor equal to 50 ng/mL. In some embodiments, the limit of detection of themethods is less than or equal to 40 ng/mL. In some embodiments, thelimit of detection of the methods is less than or equal to 35.5 ng/mL.

In some embodiments, methods provided herein comprise linearity ofquantitation across a range between 50 ng/mL to 50,000 ng/mL.

In some embodiments, methods provided herein comprise inter- andintra-assay reproducibility of CV≤15%.

In some embodiments, CgA is not derivatized prior to mass spectrometry.

In certain embodiments, the sample is a body fluid. In some embodiments,the sample is cerebrospinal fluid (CSF). In some embodiments, the sampleis plasma or serum. In some embodiments, the sample is whole blood. Insome embodiments, the sample is saliva or urine.

In some embodiments, the methods may include adding an agent to thesample in an amount sufficient to deproteinate the sample.

In some embodiments, elevated levels of chromogranin A as compared to areference range indicates increased risk of neuroendocrine tumors (NET).In some embodiments, quantitated levels of chromogranin A indicates thesize of the neuroendocrine tumor. In some embodiments, quantitatedlevels of chromogranin A indicates the tumor burden of theneuroendocrine tumor. In some embodiments, quantitated levels ofchromogranin A indicates the response to treatment of the neuroendocrinetumor. In some embodiments, quantitated levels of chromogranin Aindicates the prognosis of the neuroendocrine tumor.

Suitable test samples include any test sample that may contain theanalyte of interest. In some preferred embodiments, a sample is abiological sample; that is, a sample obtained from any biologicalsource, such as an animal, a cell culture, an organ culture, etc. Incertain preferred embodiments samples are obtained from a mammaliananimal, such as a dog, cat, horse, etc. Particularly preferred mammaliananimals are primates, most preferably male or female humans.Particularly preferred samples include blood, plasma, serum, hair,muscle, urine, saliva, tear, cerebrospinal fluid, or other tissuesample. Such samples may be obtained, for example, from a patient; thatis, a living person, male or female, presenting oneself in a clinicalsetting for diagnosis, prognosis, or treatment of a disease orcondition. The test sample is preferably obtained from a patient, forexample, blood serum.

Sample Preparation for Mass Spectrometry

Methods that may be used to enrich in CgA relative to other componentsin the sample (e.g. protein) include for example, filtration,centrifugation, thin layer chromatography (TLC), electrophoresisincluding capillary electrophoresis, affinity separations includingimmunoaffinity separations, extraction methods including ethyl acetateextraction and methanol extraction, and the use of chaotropic agents orany combination of the above or the like.

Protein precipitation is one preferred method of preparing a testsample. Such protein purification methods are well known in the art, forexample, Polson et al., Journal of Chromatography B 785:263-275 (2003),describes protein precipitation techniques suitable for use in themethods. Protein precipitation may be used to remove most of the proteinfrom the sample leaving CgA in the supernatant. The samples may becentrifuged to separate the liquid supernatant from the precipitatedproteins. The resultant supernatant may then be applied to liquidchromatography and subsequent mass spectrometry analysis. In certainembodiments, the use of protein precipitation such as for example,acetonitrile protein precipitation, obviates the need for highturbulence liquid chromatography (HTLC) or other on-line extractionprior to HPLC and mass spectrometry. Accordingly in such embodiments,the method involves (1) performing a protein precipitation of the sampleof interest; and (2) loading the supernatant directly onto the HPLC-massspectrometer without using on-line extraction or high turbulence liquidchromatography (HTLC).

In some preferred embodiments, HPLC, alone or in combination with one ormore purification methods, may be used to purify CgA prior to massspectrometry. In such embodiments samples may be extracted using an HPLCextraction cartridge which captures the analyte, then eluted andchromatographed on a second HPLC column or onto an analytical HPLCcolumn prior to ionization. Because the steps involved in thesechromatography procedures can be linked in an automated fashion, therequirement for operator involvement during the purification of theanalyte can be minimized This feature can result in savings of time andcosts, and eliminate the opportunity for operator error.

It is believed that turbulent flow, such as that provided by HTLCcolumns and methods, may enhance the rate of mass transfer, improvingseparation characteristics. HTLC columns separate components by means ofhigh chromatographic flow rates through a packed column containing rigidparticles. By employing high flow rates (e.g., 3-5 mL/min), turbulentflow occurs in the column that causes nearly complete interactionbetween the stationary phase and the analyte(s) of interest. Anadvantage of using HTLC columns is that the macromolecular build-upassociated with biological fluid matrices is avoided since the highmolecular weight species are not retained under the turbulent flowconditions. HTLC methods that combine multiple separations in oneprocedure lessen the need for lengthy sample preparation and operate ata significantly greater speed. Such methods also achieve a separationperformance superior to laminar flow (HPLC) chromatography. HTLC allowsfor direct injection of biological samples (plasma, urine, etc.). Directinjection is difficult to achieve in traditional forms of chromatographybecause denatured proteins and other biological debris quickly block theseparation columns. HTLC also allows for very low sample volume of lessthan 1 mL, preferably less than 0.5 mL, preferably less than 0.2 mL,preferably 0.1 mL.

Examples of HTLC applied to sample preparation prior to analysis by massspectrometry have been described elsewhere. See, e.g., Zimmer et al., J.Chromatogr A. 854:23-35 (1999); see also, U.S. Pat. Nos. 5,968,367;5,919,368; 5,795,469; and 5,772,874. In certain embodiments of themethod, samples are subjected to protein precipitation as describedabove prior to loading on the HTLC column; in alternative preferredembodiments, the samples may be loaded directly onto the HTLC withoutbeing subjected to protein precipitation. The HTLC extraction column ispreferably a large particle column. In various embodiments, one of moresteps of the methods may be performed in an on-line, automated fashion.For example, in one embodiment, steps (i)-(v) are performed in anon-line, automated fashion. In another, the steps of ionization anddetection are performed on-line following steps (i)-(v).

Liquid chromatography (LC) including high-performance liquidchromatography (HPLC) relies on relatively slow, laminar flowtechnology. Traditional HPLC analysis relies on column packings in whichlaminar flow of the sample through the column is the basis forseparation of the analyte of interest from the sample. The skilledartisan will understand that separation in such columns is a diffusionalprocess. HPLC has been successfully applied to the separation ofcompounds in biological samples but a significant amount of samplepreparation is required prior to the separation and subsequent analysiswith a mass spectrometer (MS), making this technique labor intensive. Inaddition, most HPLC systems do not utilize the mass spectrometer to itsfullest potential, allowing only one HPLC system to be connected to asingle MS instrument, resulting in lengthy time requirements forperforming a large number of assays.

Various methods have been described for using HPLC for sample clean-upprior to mass spectrometry analysis. See, e.g., Taylor et al.,Therapeutic Drug Monitoring 22:608-12 (2000); and Salm et al., Clin.Therapeutics 22 Supl. B:B71-B85 (2000).

One of skill in the art may select HPLC instruments and columns that aresuitable for use with CgA. The chromatographic column typically includesa medium (i.e., a packing material) to facilitate separation of chemicalmoieties (i.e., fractionation). The medium may include minute particles.The particles include a bonded surface that interacts with the variouschemical moieties to facilitate separation of the chemical moieties. Onesuitable bonded surface is a hydrophobic bonded surface such as an alkylbonded surface. Alkyl bonded surfaces may include C-4, C-8, C-12, orC-18 bonded alkyl groups, preferably C-18 bonded groups. Thechromatographic column includes an inlet port for receiving a sample andan outlet port for discharging an effluent that includes thefractionated sample. In one embodiment, the sample (or pre-purifiedsample) is applied to the column at the inlet port, eluted with asolvent or solvent mixture, and discharged at the outlet port. Differentsolvent modes may be selected for eluting the analyte(s) of interest.For example, liquid chromatography may be performed using a gradientmode, an isocratic mode, or a polytyptic (i.e. mixed) mode. Duringchromatography, the separation of materials is effected by variablessuch as choice of eluent (also known as a “mobile phase”), elution mode,gradient conditions, temperature, etc.

In certain embodiments, an analyte may be purified by applying a sampleto a column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition can be employed where the analyte of interest is retained bythe column, and a second mobile phase condition can subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. Such procedures may enrichthe amount of one or more analytes of interest relative to one or moreother components of the sample.

In one preferred embodiment, the HTLC may be followed by HPLC on ahydrophobic column chromatographic system. In certain preferredembodiments, a TurboFlow Cyclone P® polymer-based column from CohesiveTechnologies (60 μm particle size, 50×1.0 mm column dimensions, 100 Åpore size) is used. In related preferred embodiments, a SynergiPolar-RP® ether-linked phenyl, analytical column from Phenomenex Inc (4μm particle size, 150×2.0 mm column dimensions, 80 Å pore size) withhydrophilic endcapping is used. In certain preferred embodiments, HTLCand HPLC are performed using HPLC Grade Ultra Pure Water and 100%methanol as the mobile phases.

By careful selection of valves and connector plumbing, two or morechromatography columns may be connected as needed such that material ispassed from one to the next without the need for any manual steps. Inpreferred embodiments, the selection of valves and plumbing iscontrolled by a computer pre-programmed to perform the necessary steps.Most preferably, the chromatography system is also connected in such anon-line fashion to the detector system, e.g., an MS system. Thus, anoperator may place a tray of samples in an autosampler, and theremaining operations are performed under computer control, resulting inpurification and analysis of all samples selected.

In certain preferred embodiments, CgA or fragments thereof in a samplemay be purified prior to ionization. In particularly preferredembodiments the chromatography is not gas chromatography.

Detection and Quantitation by Mass Spectrometry

In various embodiments, CgA or fragments thereof may be ionized by anymethod known to the skilled artisan. Mass spectrometry is performedusing a mass spectrometer, which includes an ion source for ionizing thefractionated sample and creating charged molecules for further analysis.For example ionization of the sample may be performed by electronionization, chemical ionization, electrospray ionization (ESI), photonionization, atmospheric pressure chemical ionization (APCI),photoionization, atmospheric pressure photoionization (APPI), fast atombombardment (FAB), liquid secondary ionization (LSI), matrix assistedlaser desorption ionization (MALDI), field ionization, field desorption,thermospray/plasmaspray ionization, surface enhanced laser desorptionionization (SELDI), inductively coupled plasma (ICP) and particle beamionization. The skilled artisan will understand that the choice ofionization method may be determined based on the analyte to be measured,type of sample, the type of detector, the choice of positive versusnegative mode, etc.

In preferred embodiments, CgA or a fragment thereof is ionized byelectrospray ionization (ESI) in positive or negative mode.

After the sample has been ionized, the positively charged or negativelycharged ions thereby created may be analyzed to determine amass-to-charge ratio. Suitable analyzers for determining mass-to-chargeratios include quadrupole analyzers, ion traps analyzers, andtime-of-flight analyzers. The ions may be detected using severaldetection modes. For example, selected ions may be detected i.e., usinga selective ion monitoring mode (SIM), or alternatively, ions may bedetected using a scanning mode, e.g., multiple reaction monitoring (MRM)or selected reaction monitoring (SRM). Preferably, the mass-to-chargeratio is determined using a quadrupole analyzer. For example, in a“quadrupole” or “quadrupole ion trap” instrument, ions in an oscillatingradio frequency field experience a force proportional to the DCpotential applied between electrodes, the amplitude of the RF signal,and the mass/charge ratio. The voltage and amplitude may be selected sothat only ions having a particular mass/charge ratio travel the lengthof the quadrupole, while all other ions are deflected. Thus, quadrupoleinstruments may act as both a “mass filter” and as a “mass detector” forthe ions injected into the instrument.

One may enhance the resolution of the MS technique by employing “tandemmass spectrometry,” or “MS/MS”. In this technique, a precursor ion (alsocalled a parent ion) generated from a molecule of interest can befiltered in an MS instrument, and the precursor ion is subsequentlyfragmented to yield one or more fragment ions (also called daughter ionsor product ions) that are then analyzed in a second MS procedure. Bycareful selection of precursor ions, only ions produced by certainanalytes are passed to the fragmentation chamber, where collisions withatoms of an inert gas produce the fragment ions. Because both theprecursor and fragment ions are produced in a reproducible fashion undera given set of ionization/fragmentation conditions, the MS/MS techniquemay provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation may be used to eliminateinterfering substances, and may be particularly useful in complexsamples, such as biological samples.

The mass spectrometer typically provides the user with an ion scan; thatis, the relative abundance of each ion with a particular mass/chargeover a given range (e.g., 100 to 1000 amu). The results of an analyteassay, that is, a mass spectrum, may be related to the amount of theanalyte in the original sample by numerous methods known in the art. Forexample, given that sampling and analysis parameters are carefullycontrolled, the relative abundance of a given ion may be compared to atable that converts that relative abundance to an absolute amount of theoriginal molecule. Alternatively, molecular standards may be run withthe samples, and a standard curve constructed based on ions generatedfrom those standards. Using such a standard curve, the relativeabundance of a given ion may be converted into an absolute amount of theoriginal molecule. In certain preferred embodiments, an internalstandard is used to generate a standard curve for calculating thequantity of CgA. Methods of generating and using such standard curvesare well known in the art and one of ordinary skill is capable ofselecting an appropriate internal standard. For example, an isotope ofCgA may be used as an internal standard. Numerous other methods forrelating the amount of an ion to the amount of the original moleculewill be well known to those of ordinary skill in the art.

One or more steps of the methods may be performed using automatedmachines. In certain embodiments, one or more purification steps areperformed on-line, and more preferably all of the purification and massspectrometry steps may be performed in an on-line fashion.

In certain embodiments, such as MS/MS, where precursor ions are isolatedfor further fragmentation, collision activation dissociation is oftenused to generate the fragment ions for further detection. In CAD,precursor ions gain energy through collisions with an inert gas, andsubsequently fragment by a process referred to as “unimoleculardecomposition”. Sufficient energy must be deposited in the precursor ionso that certain bonds within the ion can be broken due to increasedvibrational energy.

In particularly preferred embodiments, CgA is detected and/or quantifiedusing MS/MS as follows. The samples are subjected to liquidchromatography, preferably HPLC, the flow of liquid solvent from thechromatographic column enters the heated nebulizer interface of an MS/MSanalyzer and the solvent/analyte mixture is converted to vapor in theheated tubing of the interface. The analyte is ionized by the selectedionizer. The ions, e.g. precursor ions, pass through the orifice of theinstrument and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 andQ3) are mass filters, allowing selection of ions (i.e., “precursor” and“fragment” ions) based on their mass to charge ratio (m/z). Quadrupole 2(Q2) is the collision cell, where ions are fragmented. The firstquadrupole of the mass spectrometer (Q1) selects for molecules with themass to charge ratios of CgA. Precursor ions with the correctmass/charge ratios of CgA are allowed to pass into the collision chamber(Q2), while unwanted ions with any other mass/charge ratio collide withthe sides of the quadrupole and are eliminated. Precursor ions enteringQ2 collide with neutral argon gas molecules and fragment. This processis called collision activated dissociation (CAD). The fragment ionsgenerated are passed into quadrupole 3 (Q3), where the fragment ions ofCgA are selected while other ions are eliminated.

The methods may involve MS/MS performed in either positive or negativeion mode. Using standard methods well known in the art, one of ordinaryskill is capable of identifying one or more fragment ions of aparticular precursor ion of CgA that may be used for selection inquadrupole 3 (Q3).

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC methods. The areas under the peaks corresponding toparticular ions, or the amplitude of such peaks, are measured and thearea or amplitude is correlated to the amount of the analyte ofinterest. In certain embodiments, the area under the curves, oramplitude of the peaks, for fragment ion(s) and/or precursor ions aremeasured to determine the amount of CgA. As described above, therelative abundance of a given ion may be converted into an absoluteamount of the original analyte, using calibration standard curves basedon peaks of one or more ions of an internal molecular standard.

The following examples serve to illustrate the invention. These examplesare in no way intended to limit the scope of the methods.

EXAMPLES Example 1: CgA Quantitation by Mass Spectrometry ReagentSummary

Reagents Supplier & Catalog Number Quantity Chromogranin-A Abcam,ab85486 250 ug Chromogranin-A IS (See New England Peptide, 6 mg CgAInternal Standard Custom Synthesis ≥95% Prep below for sequence) purityBiocell Charcoal Stripped Biocell, 1101-00 1 L and Delipidated SerumAmmonium Bicarbonate Sigma, A Millipore, 500 g (AmBic) FX0440-56141-500GTriethylammonium Sigma, T7408-500 mL 500 mL bicarbonate, 1.0MDithiothreitol Sigma, 43819-25G 25 g Iodoacetamide Sigma, I1149-25G 25 gTrypsin Sigma, T1426-500MG 0.5 g Formic Acid, 98% Millipore, FX0440-50.5 L Ammonium hydroxide, Sigma, 221228-500ML-A 500 mL 28-30% HPLC WaterBurdick & Jackson, 365-4 4 L Acetonitrile Burdick & Jackson, 015-4 4 LMethanol Fisher Scientific, A454-4 4 L Bovine Serum Albumin Sigma,A2153-500G 500 g MicroAmp Clear Thermo Fisher, 4306311 100 filmsAdhesive Film Thermo Scientific Themro, 17126-102130 1 column AccucoreC18 100 × 2.1 mm, 2.6u Oasis MAX 96-well Waters, 186000373 1 plate 30 mgSPE Plate

Sciex 6500+ QTrap Mass Spectrometer, Thermo Fisher Aria Cohesive TLX4with Agilent Pumps, Hamilton Microlab Star, SPEware IP8 were used todetect CgA.

Patient serum (100 μL) was added to 600 μL of 120 mM ammoniumbicarbonate, and the entire volume was extracted using a mixed-modeanion exchange plate. (Waters Oasis® MAX 30 mg). Samples are then washedtwice with 3% ammonium hydroxide and water, respectively. Next, CgA waseluted, internal standard (IS) added, and the sample was evaporatedunder heated nitrogen.

After dry-down, samples were reconstituted, reduced, alkylated, andtryptically digested for 2 hours in a rapid enzyme digestion microwavesystem (Hudson Technology).

Post-digestion, samples were acidified and analyzed by LC-MS/MS on anAria TLX-4 Transcend UPLC. Peptides were resolved using a ThermoAccucore C18 100×2.1 mm, 2.6 micron HPLC column usingwater/acetonitrile/0.1% formic acid gradients. The MS detector was aSciex 6500+ Qtrap. Quantitation of CgA was based on the chromatographicpeaks of fragment ions produced during MS/MS corresponding to arepresentative tryptic peptide and its corresponding heavy-labeled IS.The analytical workflow is summarized in FIG. 1.

Multiple reaction monitoring was used for detecting both the analyte andthe internal standard (IS).

Concentrations of Chromogranin A (CgA) were determined using peak arearatio and a calibration curve.

In assays detecting ELQDLALQGAK (SEQ ID NO: 1), a labeled winged peptideinternal standard was used: ILSILRHQNLLKELQDLAL*QGAK*ERAHQQK (SEQ ID NO:2): *C¹³N¹⁵ labeled amino acids. After digestion, the resulting peptidewas ELQDLAL*QGAK* (SEQ ID NO: 3).

In assays detecting RRPEDQELESLSAIEAELEK (SEQ ID NO: 4), a labeledwinged peptide internal standard was used:EGSANRRPEDQELESL*SAIEAELEK*VAHQL (SEQ ID NO: 5); *C¹³N¹⁵ labeled aminoacids. After digestion, the resulting peptide was RRPEDQELESL*SAIEAELEK*(SEQ ID NO: 6).

TABLE 1 CgA fragment used to quantitate CgA levelsin the sample and precursor and product ions (mass-to-charge m/z ratios)Ion  Precursor Ion Product Analyte (m/z) (m/z) CgA: ELQDLALQGAK 593.2516.3, 815.5 CgA IS: ELQDLAL*QGAK* 600.8 602.4, 830.6, 958.7

TABLE 2 CgA fragment used to quantitate CgA levelsin the sample and precursor and product ions (mass-to-charge m/z ratios)Ion Precursor Ion Product Analyte (m/z) (m/z) CgA: RRPEDQELESLSAIEAELEK729.6 831.5, 989.5 CgA: RRPEDQELESL*SAIEAELEK* 734.6 839.5, 997.6

TABLE 3 The values obtained were compared to the values quantitated byELISA immunoassay. See also FIG. 4. Sample ID# Average Mayo ARUP Dawn(Mayo, ARUP, Result Result Result Dawn) LCMS Ref Ranges <93 0-95 N/A N/AN/A Units ng/mL ng/mL ng/mL ng/mL ng/mL 1 1654 2286 3070 1753 1402 2 8722172 1724 1192 2084 3 1286 1603 1680 1142 1774 4 793 1151 1220 791 730 5499 1077 1285 715 827 6 588 639 657 471 4021 7 42 479 482 251 237 8 207302 297 202 589 9 277 284 285 212 677 10 156 212 187 139 412 11 114 181182 119 94 12 120 145 136 101 342 13 82 120 130 83 115 14 64 105 101 6850 15 63 96 101 65 62 16 57 88 86 58 <50 17 62 80 80 56 <50 18 47 74 7649 <50 19 41 60 60 40 <50 20 <20 36 45 40 <50

Samples from 308 patients were analyzed to compare CgA serum valuesmeasured by the Cisbio CGA-ELISA-US immunoassay (Codolet, France) withvalues from our LC-MS/MS assay.

After natural logarithm-transformation of the measurements, a normaldistribution was seen in the 308 patients. A paired t-test was performedon these data and the concordance between the assays was measured by thePearson correlation coefficient and Passing & Bablok curve fitting. Allanalyses were performed in Analyse-it v2.30.

The assay was validated. Performance characteristics are presented inTable 4:

Characteristics Values Intra-assay precision 5.2-15.7% Inter-assayprecision 7.4-10.1% Recovery of CgA spiked into 90-110% patient samplesAnalytical sensitivity    35.5 ng/mL (Limit of Detection) Analyticalsensitivity     50 ng/mL (Limit of Quantitation) Linearity 50-50,000ng/mL Reference Range <140 ng/mL (n = 160)

Typical results for patient samples with low and high concentrations ofcirculating CgA are shown in FIG. 3.

Measured CgA levels of the LC-MS/MS were on average comparable to theCisBio assay, although there was substantial scatter (Pearson'scorrelation 0.76) (FIG. 5).

Conclusion: We have developed and validated a fully automated LC-MS/MSassay for CgA from serum

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

That which is claimed is:
 1. A method for determining the amount ofchromogranin A (CgA) in a sample, said method comprising: (a) purifyingCgA in the sample; (b) ionizing CgA to produce one or more ion(s) of CgAdetectable by mass spectrometry; (c) determining the amount of theion(s) from step (b) by mass spectrometry, comprising quantifying theamount of fragment ion having a mass-to-charge ratio of 831.5±0.5 or989.5±0.5, wherein the amount of ions quantified in step (c) is relatedto the amount of CgA in the sample.
 2. The method of claim 1, whereinsaid purifying comprises extraction by solid phase extraction (SPE). 3.The method of claim 2, wherein the SPE is an anion exchange solid phaseextraction.
 4. The method of claim 2, wherein the SPE is a mixed-modeanion exchange solid phase extraction.
 5. The method of claim 2, whereinextracted samples are enzymatically digested.
 6. The method of claim 5,wherein the digestion comprises trypsin digestion.
 7. The method ofclaim 1, wherein said purifying comprises liquid chromatography.
 8. Themethod of claim 7, wherein said liquid chromatography comprises highperformance liquid chromatography (HPLC).
 9. The method of claim 7,wherein said liquid chromatography comprises high turbulence liquidchromatography (HTLC).
 10. The method of claim 1, wherein saidionization comprises electrospray ionization (ESI).
 11. The method ofclaim 1, further comprising adding an internal standard.
 12. The methodof claim 11, wherein said internal standard is isotopically labeled. 13.The method of claim 1, wherein the sample is serum.
 14. The method ofclaim 1, wherein the sample is cerebrospinal fluid (CSF).
 15. The methodof claim 1, wherein the method comprises measuring the amount ofprecursor ion having a mass-to-charge ratio of 729.6±0.5.
 16. The methodof claim 1, wherein the method comprises measuring the amount of afragment of CgA.
 17. The method of claim 16, wherein the CgA fragmentmeasured comprises a sequence RRPEDQELESLSAIEAELEK (SEQ ID NO: 4). 18.The method of claim 1, wherein the method comprises adding an internalstandard.
 19. The method of claim 18, wherein the internal standard isisotopically labeled.
 20. The method of claim 18, wherein the internalstandard comprises a C¹³N¹⁵ labeled amino acid.
 21. The method of claim18, wherein the internal standard is labeled on a leucine (L) or lysine(K).
 22. The method of claim 18, wherein the internal standard comprisesa sequence EGSANRRPEDQELESL*SAIEAELEK*VAHQL (SEQ ID NO: 5), wherein L*and K* is each a C¹³N¹⁵ labeled amino acid.
 23. The method of claim 18,wherein the method comprises measuring the amount of internal standardprecursor ion having a mass-to-charge ratio of 734.6±0.5 or product ionhaving a mass-to-charge ratio of 839.5±0.5 or 997.6±0.5.
 24. The methodof claim 1, wherein the limit of quantitation of the methods is lessthan or equal to 50 ng/mL.
 25. The method of claim 1, wherein the limitof detection of the methods is less than or equal to 35.5 ng/mL.
 26. Themethod of claim 1, wherein the method has a linearity of quantitationacross a range between 50 ng/mL to 50,000 ng/mL.
 27. The method of claim1, wherein the method has an inter- and intra-assay reproducibility ofCV≤15%.
 28. The method of claim 1, wherein the mass spectrometry isselected reaction monitoring (SRM) mass spectrometry.
 29. The method ofclaim 1, wherein the method is fully automated.
 30. The method of claim1, wherein the method is antibody-free.